Structure and Dynamic Studies of the Nuclear Pore Complex at the Single-Molecule Level

Abstract

Nuclear pore complexes (NPCs) are large macromolecular structures forming the only known direct route across the double bilayer membrane of the nuclear envelope. The NPC structure has been extensively explored in an effort to elucidate the mechanisms by which they control transport. Many of these studies have found the presence of a central mass or plug within the central channel of NPCs, although neither the function nor identity of the central mass were clear. Here, several techniques including electron microscopy, Förster resonance energy transfer (FRET), and high-resolution near-field scanning optical microscopy (NSOM) are utilized to specifically locate vault ribonucleoproteins to NPCs. This interaction, along with several other results, strongly suggests that vaults represent the central mass of NPCs. A single-molecule transport assay was also developed in order to record the translocation of individual fluorescent dextrans through NPCs. Comparison of the single-molecule dwell times under various conditions led to a better understanding of the specific mechanism controlling the non signal-mediated transport of cargo through NPCs.